CHARACTERIZATION UNIT - SUMMARY ABSTRACT The Characterization Unit will oversee collection of all image and omic data needed for construction of multi-parametric Atlases of MP lesions and CHIP specimens. Following a technology assessment/consolidation phase in year 1 most data collection will take place in the LSP and established core facilities as a means to ensure reproducibility. The Characterization Unit will work with the Analysis Unit to establish a robust information processing system that maintains the reliability of data and metadata collection and storage for Atlas specimens and QC/QA samples. Metadata will include the elements needed to comply with FAIR standards. Dissociative technologies such as single cell-RNA sequencing, DNA sequencing, and FACS/CyTOF will provide the highest dimensional data for Atlas construction but only on cells outside of their tissue context. Highly multiplexed imaging of FFPE biopsies will be used to collect spatially resolved data on cell states and morphology as well as expression of selected genes and genotypes (using padlock-rolling circle amplification probes). For the MP Atlas, laser capture of FFPE specimens will be followed by assessment of mutational status by WES, T cell clonality by TCR-seg and RNA expression by RNA-Seq. The Analysis unit will use multi-view learning and related methods to integrate the resulting spatial and omic data and to deconvolute bulk RNA sequence when scRNAseq is not possible (i.e. when analyzing FFPE MPs). Standard operating protocols developed in the Center will be evaluated and disseminated to other HTAN members. Aim 1 will characterize changes in gene expression, acquisition of mutations and cell-cell heterogeneity by sequencing, flow cytometry and CyTOF. Padlock-based FISH will be used to provide single-cell resolution of mRNA expression and mutational status. Aim 2 will collect high dimensional high resolution of images from FFPE specimens at using two complementary techniques: t-CyCIF and DEI. Antibodies will be rigorously tested and QA/QC data linked to Atlas images. A sub-set of CHIP and MP samples will be analyzed in 3D using serial sections. Aim 3 will collect time-series data from serial biopsies of CHIP patients in a longitudinal cohort and deep UMI sequencing and flow cytometry will be used to assay variant allele frequency and clonal progression. Aim 4 will implement quality assurance policies inspired by experience of quality assurance officer and pathologist Sandro Santagata in a CLIA setting. This will involve SOPs, routine retesting, competency training and statistical quality control. Aim 5 will evaluate new or improved technologies developed by PATCH Center members, other Centers in the HTAN network, collaborating investigators and companies (see letters of support). This includes direct comparison of t-CyCIF and DEI with Multiplexed Ion Beam Imaging (MIBI) now available at HMS and Scanning Mass Cytometry Imaging implemented in the Bodenmiller Lab in Zurich.